Novel peptides for preventing or treating bone diseases and use thereof

ABSTRACT

The present disclosure relates to a novel peptide for preventing or treating bone diseases. Further, the present disclosure relates to a polynucleotide encoding the peptide, a vector including the polynucleotide, a host cell transformed by the vector, and a method for producing the peptide by using the host cell. Furthermore, the present disclosure relates to a composition for preventing or treating bone diseases, including the novel peptide. The novel peptide according to the present disclosure induces mobilization of hematopoietic stem cells to blood and causes a decrease in the number of osteoclasts, and, thus, decreases bone erosion caused by osteoclasts, thereby suppressing progress of an osteoporotic lesion. Further, the novel peptide is safe since it does not cause rejection in the body. Furthermore, since the novel peptide is formed of 15 short amino acids, a low dose of the peptide can relieve symptoms of osteoporosis.

TECHNICAL FIELD

The present disclosure relates to a novel peptide for preventing ortreating bone diseases and a polynucleotide encoding the same.

Further, the present disclosure relates to a method for treating atarget with a bone disease by using the peptide.

BACKGROUND

Osteoporosis can be classified into postmenopausal osteoporosis andsenile osteoporosis. Postmenopausal osteoporosis occurs due to anincrease in bone resorption caused by activation of an osteoclastarising from a rapid hormonal change caused by menopause. Further,senile osteoporosis occurs due to a decrease in bone formation caused bya decrease in a function of an osteoblast caused by aging. Bonefractures caused by osteoporosis lead to severe restriction on activity.In particular, hip fracture is involved in high mortality of about 15 to35%. Therefore, it is important to diagnose and treat osteoporosis priorto occurrence of osteoporotic fractures.

Conventionally, bisphosphonate-based medicines have been known asmedicines for treating osteoporosis. It is known that bisphosphonatesticks to an inorganic element of bone and when an osteoclast resorbsthe bone to which bisphosphonate sticks, a non-hydrolyzed ATP analogueis formed and exhibits toxicity on the cell or causes a decrease inactivity of the osteoclast and apoptosis in various ways in theosteoclast, thereby reducing bone resorption and thus increasing a bonedensity. Although such medicines have been known as being relativelysafe, there have been recently suggested that when being used for a longtime, the medicines may affect remodeling of bone by normal boneresorption or bone formation, or healing of bone after fracture,resulting in a decrease in bone elasticity and a bad effect on bonestrength. There is a report that the medicines actually cause stressfractures in numerous patients.

Accordingly, development of novel bone metabolism involved in occurrenceof osteoporosis and development of a medicine for preventing or treatingosteoporosis has been desperately needed.

A bone marrow stem cell niche is defined as a molecular microenvironmentwhere cells' functions and fates are regulated by interactions betweenvarious cells including a bone marrow stem cell present in bone marrow.So far, there have been known roughly two kinds of bone marrow stem cellniches including “endosteal niche” and “perivascular niche”.

Firstly, “endosteal niche” refers to a microenvironment where basalcells are present in bone marrow, and basal cells, osteoblasts, andosteoclasts are mainly present. In the endosteal niche, these cells areadjacent to other cells, particularly hematopoietic progenitor cells(HPCs) or hematopoietic stem cells (HSCs) by intracellular adhesionfactors (CXCL12, Ang-1, VCAM1, stem cell factor, IL-7, and the like),and the hematopoietic stem cells sticking to the osteoblasts aremobilized to blood in response to a neurotransmission signal from theoutside or homed to the bone marrow so as to affect homeostaticmaintenance in the bone marrow.

Further, “perivascular niche” refers to a microenvironment constitutedby macrophages, bone marrow mesenchymal stem cells (MSCs), and CAR cells(CXCL12 abundant reticular cells). In the perivascular niche, thesecells are adjacent to hematopoietic cells by intracellular adhesionfactors and the hematopoietic stem cells sticking to the macrophages andthe bone marrow mesenchymal stem cells are mobilized to blood inresponse to a neurotransmission signal, such as stress or aneurotransmitter, from the outside in a similar manner to the endostealniche.

As described above, the bone marrow stem cells are mobilized to abloodstream or homed in the bone marrow niche. Mobilization of bonemarrow stem cells to blood is an important phenomenon occurring in bonemarrow when the body is stressed or damage, and refers to a phenomenonin which stem cells, immune-related cells, and osteoclasts move frombone marrow to a blood vessel. The cells mobilized to the blood vesselmove along a blood stream to a lesion site and help to heal the lesion.Meanwhile, the bone marrow stem cells that finish the healing action inthe lesion site return to the bone marrow along the blood stream, andsuch a phenomenon is referred to as “homing”.

A bone marrow hematopoietic stem cell can be differentiated intolymphoid cells and myeloid cells in bone marrow. An osteoclast as one ofthe myeloid cells differentiated from the bone marrow hematopoietic stemcell maintains bone formation and bone erosion through a balance betweenthe osteoclast and an osteoblast in the bone marrow. If such a balanceis broken, there is an increase in the number of osteoclasts and anincrease in a speed of bone erosion, resulting in occurrence ofosteoporosis.

It is known that if S1P (sphingosine-1-phosphate) as a material forinducing mobilization of osteoclast precursors to blood is administeredor an S1P receptor agonist (FTY720) is administered in order to decreasethe number of osteoclasts in bone marrow, there is an increase in boneformation. Likewise, if mobilization of bone marrow hematopoietic stemcells as primitive cells for osteoclasts to blood is induced, there is adecrease in differentiation into osteoclasts, and, thus, the number ofosteoclasts in bone marrow is decreased.

The inventors of the present disclosure have carefully tried to discovera novel active molecule which can be used for treating osteoporosis. Asa result thereof, the inventors of the present disclosure found that ashort peptide having a specific sequence induces mobilization ofhematopoietic stem cells to blood as described above and causes adecrease in the number of osteoclasts, and, thus, decreases bone erosioncaused by osteoclasts, thereby suppressing progress of an osteoporoticlesion, and denominated the peptide as “Osteopep1”.

SUMMARY

The present disclosure has been made in an effort to provide a novelpeptide for preventing or treating bone diseases.

Further, the present disclosure has been made in an effort to provide apolynucleotide encoding the peptide.

Furthermore, the present disclosure has been made in an effort toprovide a method for treating a target with a bone disease by using thepeptide.

An exemplary embodiment of the present disclosure provides a peptide forpreventing or treating a bone disease, the peptide having an amino acidsequence of SEQ ID NO: 1.

Hereinafter, the present disclosure will be described in detail.

The peptide of the present disclosure is a novel peptide having an aminoacid sequence described below, and the inventors of the presentdisclosure denominated the peptide as “Osteopep1”.

Asp-Asn-Pro-Gly-Glu-Asp-Ala-Pro-Ala-Glu-Asp-Met-Ala-Arg-Tyr (SEQ ID NO:1)

Further, the peptide of the present disclosure has the above-describedspecific sequence, and includes a peptide further including 1 to 50amino acids at an N-terminal and/or a C-terminal.

The above-described novel peptide provided in the present disclosure maybe used for preventing or treating a bone disease. The bone disease isnot limited in kind, but may be osteoporosis, osteomalacia, rickets,fibrous ostitis, an adynamic bone disease, and a metabolic bone disease,and most preferably, osteoporosis.

The novel peptide decreases an expression level of an adhesion factorfor a bone marrow hematopoietic stem cell, mobilizes the bone marrowhematopoietic stem cell in bone marrow to a bloodstream, and, thus,decreases osteoclasts in the bone marrow. Further, the peptide inducesmobilization of the hematopoietic stem cell to blood and causes adecrease in the number of osteoclasts, and, thus, decreases bone erosioncaused by osteoclasts, thereby suppressing progress of an osteoporoticlesion. Furthermore, the peptide is safe since it does not causerejection in the body. Furthermore, since the peptide is formed of 15short amino acids, a low dose of the peptide can relieve symptoms ofosteoporosis.

The peptide may be an artificially produced peptide or a recombinantpeptide.

Another exemplary embodiment of the present disclosure provides apolynucleotide encoding the peptide.

The term “polynucleotide” refers to a polymer of deoxyribonucleotide orribonucleotide that exists in a single-stranded form or adouble-stranded form. The term “polynucleotide” comprehensively includesa RNA genome sequence and a RNA sequence transcribed from a DNA (gDNAand cDNA), and unless the context clearly indicates otherwise, includesan analogue of natural polynucleotide.

The polynucleotide may include, in addition to a nucleotide sequenceencoding the peptide, a complementary sequence to the nucleotidesequence. The complementary sequence may include not only a perfectlycomplementary sequence, but also a substantially complementary sequenceto the nucleotide sequence. The complementary sequence means a sequencethat can be hybridized with a nucleotide sequence that encodes thepeptide of SEQ ID NO: 1 under stringent conditions known in the art.

Further, the polynucleotide may be changed. The change includes anaddition, a deletion, or a non-conservative substitution or aconservative substitution of a nucleotide. The polynucleotide encodingthe amino acid sequence may be interpreted as including a nucleotidesequence exhibiting a substantial identity with respect to thenucleotide sequence. The substantial identity aligns the nucleotidesequence and another random sequence in a way that they are maximallycorrespondent, and when the aligned sequence is analyzed using analgorithm generally used in the art, the sequence may exhibit greaterthan 80% identity, greater than 90% identity, or greater than 95%identity.

Furthermore, the present disclosure may provide a vector including thepolynucleotide, a host cell transformed by the vector, and a method forproducing the peptide using the host cell.

The term “vector” refers to a method for expressing a target gene in ahost cell. For example, the term “vector” includes a plasmid vector, acosmid vector, and a viral vector such as a bacteriophage vector, anadenovirus vector, a retrovirus vector, and an adeno-associated virusvector. The recombinant vector may be prepared by modifying thefollowing: a plasmid (for example, pSC101, pGV1106, pACYC177, ColE1,pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14,a series of pGEX, a series of pET, and pUC19, and the like), a phage(for example, λgt4λB, λ-Charon, λΔz1, and M13, and the like), or a virus(for example, CMV, SV40, and the like), which are often used in the art.

In the recombinant vector, the polynucleotide that encodes the aminoacid sequence of SEQ ID NO: 1 may be operatively linked to a promoter.The term “operatively linked” used herein refers to a functional bindingbetween a nucleotide expression regulatory sequence (for example, apromoter sequence) and another nucleotide sequence. Therefore, theregulatory sequence may regulate the transcription and/or translation ofthe other nucleotide sequence.

Typically, the recombinant vector may be constructed as a vector forcloning or a vector for expression. The vector for expression may be avector that is normally used in the art for expression of a foreignprotein in plants, animals, or microorganisms. The recombinant vectormay be constructed through various methods known in the art.

The recombinant vector may be constructed for use in prokaryotic oreukaryotic host cells. For example, if the recombinant vector is anexpression vector and a prokaryotic cell is used as a host cell, therecombinant vector may in general include a strong promoter fortranscription (for example, a pLλ promoter, a trp promoter, a lacpromoter, a tac promoter, a T7 promoter, and the like), a ribosomebinding site for initiating translation, and a transcription/translationtermination sequence. If a eukaryotic cell is used as a host cell, anorigin of replication operating in an eukaryotic cell included in avector may be a f1 replication origin, a SV40 replication origin, a pMB1replication origin, an adeno replication origin, an AAV replicationorigin, a CMV replication origin, a BBV replication origin, and thelike, but is not limited thereto. Further, the promoter used in therecombinant vector may be a promoter derived from a genome of a mammalcell (for example, a metallothionine promoter) or a promoter derivedfrom a virus of a mammal cell (for example, an adenovirus anaphasepromoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus (CMV) promoter, or a tk promoter of HSV), and may in generalinclude a polyadenylated sequence as a transcription terminationsequence.

Further, the present disclosure may provide a host cell transformed bythe recombinant vector.

The host cell may be any host cell known in the art. Examples of theprokaryotic cell include E. coli JM109, E. coli BL21, E. coli RR1, E.coli LE392, E. coli B, E. coli X 1776, E. coli W3110, strains ofBacillus species such as Bacillus subtillis or Bacillus thuringiensis,and intestinal bacteria and strains such as Salmonella typhymurium,Serratia marcescens, and various Pseudomonas species. Whentransformation is performed using a eukaryotic cell, a host cell may beSaccharomyce cerevisiae, an insect cell, a plant cell, or an animalcell, and may include, for example, SP2/0, CHO(Chinese hamster ovary)K1, CHO DG44, PER.C6, W138, BHK, COS-7, 293, HepG2, Huh7, 3T3, RIN, MDCKcell lines, and the like.

Furthermore, the present invention may provide a method for producing apeptide for preventing or treating bone diseases, including culturingthe host cell.

The polynucleotide or the recombinant vector including thepolynucleotide may be inserted into a host cell by using an insertionmethod widely known in the art. If the host cell is a prokaryotic cell,a CaCl₂ method and an electroporation method may be used. If the hostcell is a eukaryotic cell, a microinjection method, a calcium phosphateprecipitation method, an electroporation method, a liposome-mediatedtransfection method, or a gene bombardment method may be used. However,the method is not limited thereto. The method using a microorganism suchas E. coli has a higher productivity than that using an animal cell, butit is not suitable for production of an intact Ig antibody due toglycosylation. However, the method can be used to produce anantigen-binding fragment such as Fab and Fv.

A method for screening the transformed host cells may be carried outeasily according to a method widely known in the art by using aphenotype of a selectable marker. For example, when the selectablemarker is a resistance gene to a specific antibiotic, a transformant maybe selected easily by culturing the transformant in a medium includingthe antibiotic.

Further, yet another exemplary embodiment of the present disclosureprovides a method for treating a target with a bone disease, includingadministering a peptide having an amino acid sequence of SEQ ID NO: 1 ina therapeutically effective amount to a target in need of help.

The peptide may be administered in the form of pharmaceuticalcomposition. The peptide may be included in an amount of 0.001 to 30 wt.%, preferably 5 to 30 wt. %, and more preferably 5 to 20 wt. %, withrespect to the total weight of the pharmaceutical composition.

The pharmaceutical composition of the present disclosure may furtherinclude a suitable carrier, excipient, and diluent typically used forproduction of a pharmaceutical composition. Further, the pharmaceuticalcomposition of the present disclosure can be formulated and used in theform of oral injection such as powders, granules, tablets, capsules,suspensions, emulsions, syrups, aerosols, and the like, externalpreparations, suppositories, and sterile injection solutions accordingto a general method. Preferably, a suitable preparation known in the artmay use those described in Remington's Pharmaceutical Science (recentversion) [Mack Publishing Company, Easton Pa.]. Examples of the carrier,excipient, and diluent which can be included may include lactose,dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol,starch, acacia rubber, alginate, gelatin, calcium phosphate, calciumsilicate, cellulose, methyl cellulose, microcrystalline cellulose,polyvinyl pyrrolidone, water, methylhydroxybenzoate,propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. If thecomposition is formulated, a generally-used diluent or excipient such asa filler, an extender, a binder, a wetting agent, a disintegratingagent, a surfactant, and the like, may be used. Solid formulations fororal administration are tablets, pills, powders, granules, capsules, andthe like, and the solid formulations are prepared by mixing at least oneor more excipients, such as starch, calcium carbonate, sucrose, lactose,gelatin, and the like, into the composition. Further, not only thesimple excipients but also lubricants such as magnesium stearate andtalc may be used. Liquid formulations for oral administration aresuspensions, solutions, emulsions and syrups, and may include variousexcipients such as wetting agents, sweeteners, aromatics andpreservatives in addition to generally used simple diluents such aswater and liquid paraffin. Formulations for parenteral administrationinclude sterilized aqueous solutions, non-aqueous solvents, suspensions,emulsions, lyophilized formulations, suppositories, and the like. Thenon-aqueous solvents and suspensions may include propylene glycol,polyethylene glycol, vegetable oil such as olive oil, injectable esterlike ethylolate, and the like. A base compound of the suppositories mayinclude witepsol, macrogol, tween 61, cacao butter, laurin butter,glycerogelatin, and the like.

The term “administration” used herein refers to providing apredetermined peptide of the present disclosure or a pharmaceuticalcomposition including the peptide by a certain suitable method to asubject.

The term “subject” used herein is a subject, preferably a mammal, inneed of treatment or prevention of a bone disease. For example, thesubject may be a dog, a pig, a cow, a horse, a sheep, a rabbit, amonkey, a cat, a mouse, a rat, and the like, and may be most preferablya human.

In the treatment of the present disclosure, the peptide or thepharmaceutical composition may be administered in an amount that elicitsa biological or medicinal response in a tissue system, an animal, or ahuman that is being sought by a researcher, veterinarian, doctor orother clinician, i.e., a therapeutically effective amount that inducesrelief of symptoms of a disease or disorder to be treated. In thetreatment of the present disclosure, it is obvious to those who areskilled in the art that an effective administration amount and a numberof times of administration may be changed depending on a requiredeffect. Therefore, an optimum dose to be administered can be easilydetermined by those who are skilled in the art, and can be controlled byvarious factors, such as a type of disease, severity of disease, theamounts of an active ingredient and other components included in acomposition, a type of dosage form, and an age, a weight, a generalhealth condition, a sex, and a diet of a patient, an administrationtime, an administration route, a secretion rate of a composition, atreatment period, a drug of simultaneous use, and the like. For apreferable effect, the peptide or the composition of the presentdisclosure may be administered in an amount of 0.05 to 0.1 mg/kg/day,preferably 0.01 to 0.1 mg/kg/day, and may be administered one time orseveral times per day.

In the treatment of the present disclosure, the peptide or thecomposition may be administered to the subject through various routes.All of administration methods are expectable. For example, theadministration method may be carried out by oral dosage, rectaladministration, or intravenous injection, intramuscular injection,hypodermic injection, intradural injection within the womb, orcerebrovascular injection.

Further, the treatment of the present disclosure may be used alone forpreventing or treating bone diseases, or may be used in combination withsurgery, radiation therapy, hormonal therapy, chemotherapy, or othermethods using a biological response regulator.

Further, the peptide having an amino acid of SEQ ID NO: 1 of the presentdisclosure may be added to food in order to be used for preventing orimproving bone diseases. If the peptide of the present disclosure isused as a food additive, the peptide may be appropriately used accordingto generally used methods by being added as it is or being mixed withother food or food ingredients.

The peptide as an active component may be appropriately changed in anamount depending on a usage purpose (prevention, health, or therapeutictreatment), and may be included in an amount of 0.001 to 30 wt. %,preferably 5 to 30 wt. %, and more preferably 5 to 20 wt. %, withrespect to the total weight of food ingredients.

To be specific, for example, when food or beverage is produced, thepeptide of the present disclosure may be added in an amount of 15 wt. %or less, preferably 10 wt. % or less, with respect to the material.However, for a long-time ingestion with a purpose of promoting health,hygiene or regulating health, the amount of the peptide may be less thanthe above-described range. Otherwise, since the peptide has no problemin view of safety, the amount may be greater than the above describedrange.

There is no specific limitation in the kind of the food. Examples of thefood to be added with the peptide may include meat, sausage, bread,chocolate, candies, snacks, sweets, pizza, ramen, other noodles, gums,dairy foods including ice cream, various kinds of soups, beverages,teas, drink preparations, alcoholic beverages, and vitamin complexes,and may include all kinds of health functional food in an acceptedmeaning.

If a beverage including the peptide is produced, the beverage mayinclude additional ingredient such as various flavoring agents ornatural carbohydrates like general beverages. Examples of the naturalcarbohydrates include monosaccharides such as glucose and fructose;disaccharides such as maltose and sucrose; natural sweeteners such asdextrin and cyclodextrin, and synthetic sweeteners such as saccharin,aspartame, and the like.

In addition, the food for preventing or improving bone diseases of thepresent disclosure may include various nutritional supplements,vitamins, electrolytes, flavoring agents, colorants, pectic acid and itssalt, alginic acid and its salt, organic acids, protective colloidalthickeners, pH regulating agents, stabilizers, preservatives, glycerin,alcohols, carbonizing agents used in carbonated drinks, and the like.Moreover, the food of the present disclosure may include fruit flesh forpreparation of natural fruit juices, fruit juice beverages, andvegetable beverages. These ingredients may be used alone or incombination. Although a ratio of the additives is not strictly limited,these additives are generally included in an amount of 0.01 to 0.1 wt. %with respect to the total weight of the food of the present disclosure.

According to the exemplary embodiments of the present disclosure, thenovel peptide of the present disclosure can induce mobilization of thehematopoietic stem cell to blood and cause a decrease in the number ofosteoclasts, and, thus, decrease bone erosion caused by osteoclasts,thereby suppressing progress of an osteoporotic lesion. Further, thepeptide of the present disclosure is safe since it does not causerejection in the body. Furthermore, since the peptide is formed of 15short amino acids, a low dose of the peptide can relieve symptoms ofosteoporosis.

The foregoing summary is illustrative only and is not intended to be inany way limiting. In addition to the illustrative aspects, embodiments,and features described above, further aspects, embodiments, and featureswill become apparent by reference to the drawings and the followingdetailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram illustrating a change in an expression level of anadhesion factor for a bone marrow hematopoietic stem cell when Osteopep1of the present disclosure is administered.

FIG. 2 is a diagram illustrating the number of bone marrow hematopoieticprogenitor cells in blood when Osteopep1 of the present disclosure isadministered.

FIG. 3 is a diagram illustrating a mobilization level of Osteopep1 ofthe present disclosure to blood by using a marker of a bone marrowhematopoietic stem cell.

FIG. 4 illustrates an overview of an experiment conducted to find out aneffect of Osteopep1 of the present disclosure on an expression level ofan adhesion factor for a bone marrow hematopoietic stem cell.

FIG. 5 illustrates a result of an effect of Osteopep1 of the presentdisclosure on an expression level of an adhesion factor for a bonemarrow hematopoietic stem cell.

FIG. 6 illustrates an overview of an experiment conducted to find out aneffect of Osteopep1 of the present disclosure on osteoporosis andtreatment thereof.

FIG. 7 is a diagram illustrating a change in an expression level of anadhesion factor for a bone marrow hematopoietic stem cell when Osteopep1is administered to an osteoporosis model.

FIG. 8 is a diagram illustrating the number of bone marrow hematopoieticprogenitor cells in blood when Osteopep1 is administered to anosteoporosis model.

FIG. 9 is a diagram illustrating a mobilization level of bone marrowhematopoietic stem cells to blood when Osteopep1 is administered to anosteoporosis model, by using a marker of a bone marrow hematopoieticstem cell.

FIG. 10 provides micro CT images exhibiting an overall change in bonedensity when Osteopep1 is administered to an osteoporosis model.

FIG. 11 provides graphs quantifying a change in bone density and achange in trabecular thickness, which are measured by micro CT, whenOsteopep1 is administered to an osteoporosis model.

DETAILED DESCRIPTION

In the following detailed description, reference is made to theaccompanying drawing, which forms a part hereof. The illustrativeembodiments described in the detailed description, drawing, and claimsare not meant to be limiting. Other embodiments may be utilized, andother changes may be made, without departing from the spirit or scope ofthe subject matter presented here.

Hereinafter, the present disclosure will be described in more detailwith reference to Examples. However, Examples are provided forillustrative purposes only and not intended to limit the scope of thepresent disclosure.

Example 1. Materials for Experiment and Experiment Method

1-1. Preparation of Mice and Protocol of Drug Treatment

All of the mice used in the experiment were 6-week- to 8-week-oldC57BL/6 mice and purchase from Jackson Laboratory (Bar Harbor, Me.,USA).

Osteopep1 used herein was produced by PEPTRON. For in vitro experiment,0 nM Osteopep1 and 10 nM Osteopep1 were diluted in respective media andthen injected. Further, for in vivo experiment, three mice per group,fifteen mice in total, were anesthetized with a mixed solution of 100mg/kg of ketamine and 10 mg/kg of xylazine. Further, the each mouse wasadministered with 50 μg/kg of Osteopep1 and 100 μl of PBS (Gibco) byintravenous injection to its tail.

In order to prepare osteoporosis models, three 12-week-old female miceper group, six 12-week-old female mice in total, had an ovariectomy.After 1 week, 50 μg/kg of Osteopep1 (PEPTRON) and 100 μl of PBS (Gibco)were intraperitoneally administered every 12 hours twice per day for 3weeks. For control groups as sham osteoporosis models, three female miceper group, six female mice in total, had a subcision.

1-2. Culturing of Bone Marrow Mesenchymal Stem Cell and Induction ofDifferentiation into Osteoblast

After a 4-week- to 6-week-old C57BL/6 mouse was anesthetized andsacrificed, the tibias and the femurs were removed. Bone marrow washarvested from the tibias and the femurs, and a single cell suspensionwas obtained by using a 40 μm cell strainer (Becton-Dickinson LAβware,Franklin Lakes, N.J.). About 10⁷ cells were divided in a 75-cm² flaskincluding Mesenchymal Stem Cell Stimulatory Supplements (Stem CellTechnologies, Inc.) added with antibiotics and MesenCult™ MSC Basalmedium. After being cultured for one week, the cells were cultured forthree weeks in StemXVivo Osteogenic/Adipogenic Base Media (R&D systems)added with StemXVivo Osteogenic supplement (20×) andpenicillin-streptomycin (100×) for differentiation into osteoblasts. Theculture media were replaced every 2 to 3 days.

1-3. Real-Time Quantitative PCR

In order to measure expression levels of adhesion factors (Sdf-1a, Kit1,Angpt1, IL7, Vcam1, Spp1) for hematopoietic stem cells present inosteoblasts, a real-time quantitative PCR was used.

An RNeasy Plus mini kit (Qiagen, Korea, Ltd.) was used to extract totalRNA from a cell eluent and bone marrow cells. A kit produced by Clontech(Mountain View, Calif.) was used to synthesize cDNA from 5 μg of thetotal RNA. Further, a Corbett research RG-6000 real-time PCR device wasused to perform a real-time quantitative PCR repeatedly for 40 cyclesunder a condition of 95° C. for 10 minutes; 95° C. for 10 seconds; 58°C. for 15 seconds; and 72° C. for 20 seconds per cycle.

Primers used in the real-time quantitative PCR were as listed in thefollowing Table.

TABLE 1 SDF-1 α F 5′-TTCCTATCAGAGCCCATAGAG-3′ R5′-CCAGACCATCCTGGATAATG-3′ Kit ligand (stem cell F5′-CCAAAAGCAAAGCCAATTACAAG-3′ factor; SCF) R 5′-AGACTCGGGCCTACAATGGA-3′Angiopoietin-1 F 5′-ACGGGGGTCAATTCTAAG-3′ (Angpt1) R5′-GCCATTCCTGACTCCACA-3′ Vascular cell adhesion F5′-AAAAGCGGAGACAGGAGACA-3′ molecule-1 (Vcam1) R5′-AGCACGAGAAGCTCAGGAGA-3′ IL7 F 5′-ATTGAACCTGCAGACCAAGC-3′ R5′-GCAACAGAACAAGGATCAGG-3′ Spp1 (osteopontin)  F5′-TGTGGAGTTTTAGAGATATTAGATAGTGGG-3′ R5′-AACACACTCTTAACACCACTAAATCACC-3′ GAPDH F 5′-TTGCTGTTGAAGTCGCAGGAG-3′ R5′-TGTGTCCGTCGTGGATCTGA-3′

1-4. Colony-Forming Unit (CFU) Assays

In order to measure the number of bone marrow hematopoietic progenitorcells in blood of a mouse, CFU assays were conducted.

A mouse was anesthetized, and then, 500 μl to 700 μl of blood wascollected from the heart into a heparin tube. Then, the collected bloodwas put into an ammonium chloride solution (Stem Cell Technologies, Inc.1:10) and then placed in ice for 15 minutes, and red blood cells wereremoved. The resultant solution was shaken every 2 to 3 minutes toremove red blood cells well, and then, centrifuged for 7 minutes at 1000rpm. The supernatant was removed, and the resultant solution was washedwith IMDM (Gibco) supplied with 2% fetal bovine serum (FBS) (Gibco). Thewashed cells (3×10⁵ per mouse) were divided into three 35 mm dishes(1×10⁵ per dish) respectively including methylcellulose-based media(Methocult, Stem cell), and then, cultured for 2 weeks. Then, the numberof colonies in the flask was counted.

1-5. Flow Cytometry Analysis (FACs)

In order to find out any change in the number of bone marrowhematopoietic stem cells present in bone marrow of mice, Osteopep1 andPBS were injected to normal mice, respectively. After 60 minutes, bonemarrow was harvested, and FACs was conducted to the harvested bonemarrow by using three kinds of antibodies including Lineage, Sca-1, andc-kit as markers of bone marrow hematopoietic stem cells.

For analyzing bone marrow hematopoietic stem cells, red blood cells wereremoved from bone marrow, which was harvested from the tibias and thefemurs of a 4-week- to 6-week-old C57BL/6 mouse, with an ammoniumchloride solution (Stem Cell Technologies, Inc. 1:4) and then, the bonemarrow was washed with a PBS (Gibco) solution including 10% fetal bovineserum (FBS) (Gibco) and 1% sodium azide (Sigma-Aldrich) and centrifugedfor 10 minutes at 300×g. The hematopoietic cells included in the bonemarrow were removed with MACs beads (Miltenyi Biotec) by using abiotinylated lineage antibody (Miltenyi Biotec), and the remaining cellswere reacted at 4° C. for 30 minutes by using Sca-1-PECY7, c-kit-APC,CD150-PE, and CD48-FITC antibodies (BD science) and then analyzed with aflow cytometer LSRII (BD science).

1-6. Micro CT

The femurs was separated from the mouse and refrigerated in 80% ethanol.Then, for micro CT scanning, a tissue having a thickness of 40 μm wasmeasured with a micro CT scanner (Inveon preclinical CT, SiemensHealthcare, Hoffman Estates, Ill.) under conditions including anexposure time of 600 msec, photon energy of 70 keV, and a current of 400μA. In order to measure a bone density (bone volume/total volume) and atrabecular thickness, pieces each having a volume of 2.5×0.5×0.5 mm³from the same site of each group were measured with Siemens InveonSoftware.

Example 2. Effect of Osteopep1 on Expression of Adhesion Factor for

bone marrow hematopoietic stem cell and number of hematopoietic stemcells in blood

In order to find out an effect of Osteopep1 on expression of adhesionfactors (Sdf-1a, Kit1, IL7, Vcam1, Spp1) for hematopoietic stem cellspresent in osteoblasts and the number of bone marrow hematopoieticprogenitor cells in blood, the following experiment was conducted.

2-1. Expression Level of Adhesion Factor for Hematopoietic Stem Cell

In order to find out in vivo effects of Osteopep1 of the presentdisclosure on an expression level of an adhesion factor for a bonemarrow hematopoietic stem cell, a mouse was administered with 50 μg/kgof Osteopep1 by intravenous injection to its tail, and after 60 minutes,bone marrow was harvested from the tibia and the femur of the mouse.Then, expression levels of the adhesion factors were checked by areal-time quantitative PCR.

The result thereof was as illustrated in FIG. 1.

As illustrated in FIG. 1, the expression levels of the main adhesionfactors Sdf-1a, Kit1, Angpt1, and Spp1 were decreased (p<0.05, n=3 pergroup).

2-2. Measurement of Number of Bone Marrow Hematopoietic Progenitor Cellsin Blood (CFU Assay)

In order to check whether mobilization of bone marrow hematopoieticprogenitor cells to blood is induced by a decrease in expression levelof the adhesion factors caused by administration of Osteopep1 of thepresent disclosure, a mouse was administered with 50 μg/kg of Osteopep1by intravenous injection to its tail, and after 60 minutes, blood wascollected from the heart. Then, a CFU (Colony-forming unit) assay wasconducted thereto.

The result thereof was as illustrated in FIG. 2.

As illustrated in FIG. 2, it could be seen that administration ofOsteopep1 increased mobilization of bone marrow hematopoietic progenitorcells to blood (p<0.05, n=3 per group).

2-3. Measurement of Number of Bone Marrow Hematopoietic Stem Cells inBone Marrow (FACs Assay)

In order to find out effects of administration of Osteopep1 of thepresent disclosure on the number of bone marrow hematopoietic stem cellspresent in bone marrow, the following experiment was conducted accordingto the method of Example 1-5.

Firstly, normal mice were divided into two groups each including 3 mice,and the respective groups were administered with 50 μg/kg of Osteopep1and 100 μl of PBS (Gibco). After 60 minutes, bone marrow was harvested,and FACs was conducted to the harvested bone marrow by using three kindsof antibodies including Lineage, Sca-1, and c-kit as markers of bonemarrow hematopoietic stem cells.

The result thereof was as illustrated in FIG. 3.

As illustrated in FIG. 3, the bone marrow hematopoietic stem cellsmarked by Lineage, Sca-1, and c-kit were decreased by administration ofOsteopep1 rather than administration of PBS (p<0.05, n=3 per group).

Example 3. Effect of Osteopep1 of the Present Disclosure on Expressionof Adhesion Factor for Hematopoietic Stem Cell

In order to find out effects of Osteopep1 on an expression level of anadhesion factor involved in maintaining of bone marrow hematopoieticstem cell within bone marrow, the following experiment was conductedaccording to the method of Example 1-2 and Example 1-3.

Firstly, bone marrow was harvested from a 4-week- to 6-week-old C57BL/6mouse, and bone marrow mesenchymal stem cells (BM-MSC) were collectedand cultured for 3 weeks in bone cell differentiation-inducing mediumand thus differentiated into osteoblasts. On the 21st day, theosteoblasts were treated with 0 nM Osteopep1 and 10 nM Osteopep1 forthree days. Then, bone cells were collected, and expression levels ofsix kinds of adhesion factors were checked by a real-time quantitativePCR.

A mimetic diagram relevant to the experiment process was as illustratedin FIG. 4, and the measurement result of the expression levels of theadhesion factors was as illustrated in FIG. 5.

As illustrated in FIG. 5, it could be seen that as compared with theosteoblasts (Control) which were not treated with Osteopep1, theexpression levels of Sdf-1a, Kit1, and Angpt1 in the osteoblasts treatedwith 0 nM Osteopep1 and 10 nM were concentration-dependently decreased(p<0.05, n=3 per group).

It could be seen from the above result that Osteopep1 decreased anexpression level of an adhesion factor for a bone marrow hematopoieticstem cell present in an osteoblast, and, thus, mobilization of bonemarrow hematopoietic stem cells to blood from bone marrow could beinduced.

Example 4. Effect of Osteopep1 of the Present Disclosure on Preventionand Treatment of Osteoporosis

In order to check whether mobilization of a hematopoietic stem cell toblood caused by administration of Osteopep1 of the present disclosurehas an effect on prevention and treatment of osteoporosis, the followingexperiment was conducted according to the method of Example 1-1 andExample 1-6.

In order to prepare osteoporosis models, three 12-week-old female miceper group had an ovariectomy. After 1 week, 50 μg/kg of Osteopep1(PEPTRON) and 100 μl of PBS (Gibco) were intraperitoneally administeredevery 12 hours twice per day for 3 weeks. For a control group, a normalmouse had a subcision and was intraperitoneally administered with 100 μlof PBS or 50 μg/kg of Osteopep1. On the 22nd day, the femurs wasseparated from the mouse and refrigerated in 80% ethanol. Then, a bonedensity and a trabecular thickness were measured by micro CT.

4-1. Effect of Osteopep1 on Expression of Adhesion Factor for BoneMarrow Hematopoietic Stem Cell in Osteoporosis Model

In order to find out effects of Osteopep1 on an expression level of anadhesion factor involved in maintaining of bone marrow hematopoieticstem cell within bone marrow in an osteoporosis model, the followingexperiment was conducted according to the method of Example 1-3.

A mimetic diagram relevant to the experiment process was as illustratedin FIG. 6, and the result thereof was as illustrated in FIG. 7.

As illustrated in FIG. 7, when Osteopep1 was administered, theexpression levels of the adhesion factors (Sdf-1a, Kit1, Angpt1, IL7,Vcam1, Spp1) for hematopoietic stem cells in the osteoporosis modelswere decreased (p<0.05, n=3 per group).

4-2. Effect of Osteopep1 on Mobilization of Bone Marrow HematopoieticProgenitor Cell to Blood in Osteoporosis Model

In order to find out effects of administration of Osteopep1 of thepresent disclosure on bone marrow hematopoietic progenitor cellsmobilized to blood in an osteoporosis model, an experiment was conductedin the same manner as the method of Example 1-4.

The result thereof was as illustrated in FIG. 8.

As illustrated in FIG. 8, it could be seen that Osteopep1 increasedmobilization of bone marrow hematopoietic progenitor cells to blood inthe osteoporosis models (p<0.05, n=3 per group).

4-3. Effect of Osteopep1 on Mobilization of Bone Marrow HematopoieticProgenitor Cell to Blood in Osteoporosis Model and Micro CT

In order to check whether mobilization of bone marrow hematopoieticprogenitor cells to blood in an osteoporosis model is induced byOsteopep1 of the present disclosure, the following experiment wasconducted.

The result thereof was as illustrated in FIG. 9.

As illustrated in FIG. 9, the mobilized amounts of the markers of bonemarrow hematopoietic stem cells were increased (p<0.05, n=3 per group).

It could be seen from the above result that a long-time administrationof Osteopep1 of the present disclosure decreased an expression level ofan adhesion factor for a bone marrow hematopoietic stem cell present inan osteoblast, and, thus, mobilization of bone marrow hematopoietic stemcells to blood could be induced.

FIG. 10 provides micro CT images showing bone of a control group and anosteoporosis model, and FIG. 11 provides graphs exhibiting changes inbone density and trabecular thickness.

As shown in FIG. 10, it could be seen from the micro CT images that abone density percentage (BV/TV, %) and a trabecular thickness (mm) ofthe osteoporosis model mouse injected with Osteopep1 were increased ascompared with the osteoporosis model mouse injected with PBS, and asimilar tendency was observed in the control group models (n=3 pergroup).

It can be seen from the above result that mobilization of bone marrowhematopoietic stem cells to blood caused by a long-time administrationof Osteopep1 to a mouse with osteoporosis induces differentiation from ahematopoietic stem cell into osteoclasts and a decrease in the number ofosteoclasts within bone marrow, and, thus, it is possible to suppressdecreases in bone density and trabecular thickness in the mouse withosteoporosis.

From the foregoing, it will be appreciated that various embodiments ofthe present disclosure have been described herein for purposes ofillustration, and that various modifications may be made withoutdeparting from the scope and spirit of the present disclosure.Accordingly, the various embodiments disclosed herein are not intendedto be limiting, with the true scope and spirit being indicated by thefollowing claims.

1. A peptide comprising the amino acid sequence of SEQ ID NO:
 1. 2. (canceled)
 3. The peptide of claim 1, wherein the peptide decreases an expression level of an adhesion factor for a bone marrow hematopoietic stem cell and mobilizes the bone marrow hematopoietic stem cell in bone marrow to a bloodstream.
 4. The peptide of claim 1, wherein the peptide decreases osteoclasts in the bone marrow.
 5. A polynucleotide encoding a peptide of claim
 1. 6-9. (canceled)
 10. The peptide of claim 1 that consists of the amino acid sequence set forth in SEQ ID NO:
 1. 11. A pharmaceutical composition comprising the peptide of claim 1 and a pharmaceutically acceptable carrier, excipient, and diluent.
 12. The pharmaceutical composition of claim 10, that is formulated for oral administration.
 13. The pharmaceutical composition of claim 10, that is formulated for parenteral administration. 